In a perfusion culture of suspension cells (perfusion meaning a continuous flow of culture medium through a culture maintained at high cell density; suspension cells being cells which do not require a solid support to grow on), for example in the production of monoclonal antibodies or viral vaccines, fresh nutrients must be supplied continuously with concomitant removal of toxic metabolites and, ideally, selective removal of dead cells. Removing spent medium while retaining cells in a bioreactor presents a technical problem due to the small size of cells and an average cell density only slightly greater than that of the culture medium. Filtering, entrapment and micro-capsulation methods are all suitable for refreshing the culture environment at sufficient rates. However, these methods all share certain characteristic disadvantages, chief among which are accumulation of dead cells within the system and membrane bio-fouling, which hinder attempts at industrial-scale application. The above disadvantages have a considerable impact on the quantitative recovery of active product.
Known systems for selective removal of dead cells during perfusion culture are, with very few exceptions, based on cell sedimentation. For the reasons mentioned above (i.e. small cell size and equally small density difference between cells and culture medium), unit gravity is rarely considered sufficient. The effect of gravity is multiplied, either by the use of a centrifuge, or by exploiting the Boycott effect. This effect, quantified in 1920, is a finding of faster settling of suspended particles between closely spaced inclined surfaces than in vertical chambers.
In many cases, dead suspension cells have a smaller cell diameter than live cells of the same cell line. This difference in diameter translates into a proportional difference in sedimentation rate, which can be exploited for the selective removal of dead cells.
Certain existing sedimentation systems are based on laminar flow in a conical sedimenter while some others exploit so-called lamellar settlers. In most cases, the latter feature a series of parallel plates, inclined most often at 30.degree. from the vertical, within a rectangular chamber. As fluid flows upwards between the parallel plates, particles settle onto the upper surfaces of the plates and slide toward the bottom of the chamber for collection.
B. C. Batt et al., Biotechnol. Prog., 6, 458-464, (1990) describe a continuous perfusion system consisting of a standard stirred-tank laboratory-scale bioreactor with a rectangular lamellar settler disposed above the former. Sato et al., J. Tiss. Cult. Meth. 8(4), pp. 167-171, 1983, developed an internal conical cell separator in which a tapered conical sedimentation chamber was affixed to the underside of the headplate of the bioreactor vessel.
A recently developed conical lamellar settler (M. Tyo, presentation at the 1989 annual AlChE meeting in Santa Barbara, Calif.; New Developments in Mammalian Cell Reactor Studies, Nov. 5-10, 1989), combines the advantages of conical and lamellar settlers. A conical settler is affixed to the top of a standard 250 ml spinner flask. The settler consists of a pair of concentric truncated cones with an innermost solid cone. As the cell suspension is pumped upwards from the reactor into and through the settler, the latter creates an annular flow passageway of increasing cross-sectional area with increasing distance from the lower apex. As a result, the vertical component of the liquid velocity decreases, which allows the sedimentation of cells and their collection on the upper surfaces of the truncated cones. Sedimented cells slide along these surfaces by gravity and return to the reactor, while partially clarified cell suspension (containing cells too small to have sedimented) is removed through the top of the settler. Because the settler has to occupy the entire available area at the top of the cylindrical reactor vessel, the scale-up of this design is very limited.
Hamamoto et al, U.S. Pat. No. 4,814,278 issued Mar. 21, 1989, describes an apparatus for perfusion cell culture, which has a cell culture zone and an annular cell settling zone disposed concentrically around the culture zone and separated therefrom by a partition allowing the two zones to communicate only below the partition. At any scale larger than a few liters, this design imposes aspect ratios in the culture zone which are unsuitable for the culture of animal cells. Thus the scale-up of this design is also severely limited.
There is still a need to develop a system which could offer, beside an efficiency at least comparable to known systems, a possibility of scale-up and a simpler design.